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Abstract

Pendrin is an anion exchanger expressed along the apical plasma membrane and apical cytoplasmic vesicles of type B and of non-A, non-B intercalated cells of the distal convoluted tubule, connecting tubule, and cortical collecting duct. Thus, Pds (Slc26a4) is a candidate gene for the putative apical anion-exchange process of the type B intercalated cell. Because apical anion exchange–mediated transport is upregulated with deoxycorticosterone pivalate (DOCP), we tested whether Pds mRNA and protein expression in mouse kidney were upregulated after administration of this aldosterone analogue by using quantitative real-time polymerase chain reaction as well as light and electron microscopic immunolocalization. In kidneys from DOCP-treated mice, Pds mRNA increased 60%, whereas pendrin protein expression in the apical plasma membrane increased 2-fold in non-A, non-B intercalated cells and increased 6-fold in type B cells. Because pendrin transports HCO3− and Cl−, we tested whether DOCP treatment unmasks abnormalities in acid-base or NaCl balance in Pds (-/-) mice. In the absence of DOCP, arterial pH, systolic blood pressure, and body weight were similar in Pds (+/+) and Pds (-/-) mice. After DOCP treatment, weight gain and hypertension were observed in Pds (+/+) but not in Pds (-/-) mice. Moreover, after DOCP administration, metabolic alkalosis was more severe in Pds (-/-) than Pds (+/+) mice. We conclude that pendrin is upregulated with aldosterone analogues and is critical in the pathogenesis of mineralocorticoid-induced hypertension and metabolic alkalosis.

Along the renal collecting duct, secretion of acid or base occurs through intercalated cells. Whether an intercalated cell secretes or absorbs net H+ equivalents depends at least in part on whether the H+-ATPase localizes to the apical or the basolateral plasma membrane.1 Therefore, intercalated cells are also subclassified on the basis of the presence or absence of the anion exchanger, AE1, and the distribution of the H+-ATPase within the cell.2–5 Each of these intercalated cell subtypes can be identified on the basis of ultrastructural characteristics.5 Type A intercalated cells are believed to secrete H+ equivalents. However, the physiologic role of non-A, non-B intercalated cells in acid-base homeostasis is unknown.5

Rodents can ingest a substantial base load and yet develop only a mild elevation in serum HCO3−.6 The cortical collecting duct (CCD) is a critical component of the kidney’s robust ability to excrete OH− equivalents during metabolic alkalosis. In the CCD, secretion of OH− equivalents occurs in large part through the type B intercalated cell,4 wherein Cl−/HCO3− exchange across the apical plasma membrane functions in series with the H+-ATPase expressed on the basolateral plasma membrane.2,3,5 The gene product(s) responsible for this apical anion exchange–mediated HCO3− secretion has been a matter of controversy. However, recent evidence points to the Na+-independent Cl−/HCO3− exchanger Pds7,8 as a candidate gene product.

Everett and colleagues9 first reported the molecular structure of the gene disrupted in the Pendred syndrome (Pds). In the kidney, pendrin is expressed in the CCD, the connecting tubule (CNT), the initial collecting tubule (iCT), and the distal convoluted tubule (DCT).7,10,11 Within these segments, pendrin localizes to the apical plasma membrane and apical cytoplasmic vesicles of both type B and non-A, non-B intercalated cells.7,10,11 Although pendrin is highly expressed in kidney, both Pds-knockout mice (Pds-/-) and individuals with genetic disruption of the Pds gene (Pendred syndrome) have normal renal function and normal acid-base, fluid, and electrolyte balance.7 However, a renal phenotype might be unmasked in Pds(-/-) mice under conditions that upregulate Pds in normal animals.

Whether Pds expression changes in tandem with activity of the putative apical anion exchanger is now a subject of intense interest. Both apical anion-exchange activity and pendrin protein expression are upregulated after NaHCO3 ingestion.12–14 Aldosterone administration represents another model of metabolic alkalosis in which apical anion exchange is upregulated.15,16 However, the effect of aldosterone on Pds expression is unknown.

We therefore asked 3 questions: first, are Pds mRNA and protein upregulated in another model of metabolic alkalosis, ie, administration of an aldosterone analogue (deoxycorticosterone pivalate [DOCP])? Second, is the subcellular distribution of pendrin altered with DOCP administration? and finally, is a renal phenotype, such as an acid-base abnormality or dysregulation of fluid and/or electrolyte balance, unmasked with DOCP administration in Pds(-/-) mice?

Methods

Animals

Series 1

Male, non-Swiss albino mice (Harlan, Ardmore, Tex) weighing 20 to 30 g were fed a balanced 0.07% Na+, 0.8% K+ diet (53881300 Zeigler Brothers) prepared as a gel. Control and treated mice, chosen at random, were given 18 g of a gelled, 0.6% agar diet for 6 days before sacrifice. Mice received 13.5 mL water and 4.5 g mouse chow supplemented with 0.7 mEq NaCl per day added to the gel. Treated mice received 1.7 mg DOCP (Ciba-Geigy Animal Health) by intramuscular injection 7 days before sacrifice.

Series 3

Pds(-/-) and Pds (+/+) mice, fed as in series 2, received 1.7 mg DOCP IM 7 days before sacrifice. Mice were placed in metabolic cages, and urine was collected on ice under oil for 24 hours before sacrifice. All mice were anesthetized with 4% isoflurane in 100% O2 at 1 L/min before sacrifice. The Institutional Animal Care and Use Committee at Emory and the University of Texas Health Science Center (UTHSC) approved all animal treatment protocols.

Systolic blood pressure in conscious mice was measured by the tail-cuff method with use of a MOD 59 pulse amplifier (Innovators in Instrumentation) or BP-2000 (Visitech Systems). Blood was collected for serum chemistry analyses by cardiac puncture under isoflurane anesthesia. Unless noted, urine and serum chemistry values were measured with commercially available instruments (Hitachi 717 and 747 analyzers) at IDEXX Laboratories (West Sacramento, Calif). For arterial blood gases and serum K+, mice were anesthetized with isoflurane for 15 minutes before sample collection. An abdominal incision was made, and 0.15 mL blood was drawn into a heparinized syringe through the abdominal aorta. The sample was placed on ice, and arterial blood gases and K+ were measured immediately at the Hermann Hospital Clinical Laboratory on a commercially available instrument (IL 1620, Instrumentation Laboratories, or an AVL OPTI 1 Blood Gas Analyzer, AVL Medical Instruments). Urinary pH was measured immediately after collection of urine into a gas-tight syringe by bladder puncture of anesthetized mice. Urine osmolality was measured with a vapor pressure osmometer (Wescor). Urinary total ammonia concentration was measured on a continuous-flow fluorimeter18 and a kit (171-A, Sigma Chemical). Net acid excretion was taken as the sum of the 24-hour urinary excretion of total ammonia concentration plus titratable acid. Urinary HCO3− concentration was assumed to be zero. Titratable acid was calculated by using the measured urinary pH and phosphorous concentration and a pK for phosphate of 6.8.19

Preparation of Total RNA and Quantitative Real-Time RT-PCR

Total RNA was isolated from mouse kidney by using a mini-kit (Qiagen Rneasy, Qiagen).10 Quantitative real-time polymerase chain reaction (PCR) was performed in the Quantitative Genomics Core Laboratory in the Department of Integrative Biology and Pharmacology, UTHSC, with use of a sequence detector (7700 sequence detector, Applied Biosystems).10 Specific quantitative assays for mouse Pds and β-actin were used.10 β-Actin and Pds mRNAs were measured in the same samples and expressed per left kidney.

Pendrin Immunolocalization

Kidneys were preserved and processed for light and electron microscopy as described previously.10 For light microscopy, pendrin immunoreactivity was localized by routine immunoperoxidase techniques.10 For electron microscopy, pendrin immunoreactivity was localized in ultrathin sections by immunogold cytochemistry.10 The CCDs, connecting segments (CNT), and initial collecting tubules (iCT) were identified as described previously.10 Type A, type B, and non-A, non-B intercalated cell subtypes were identified by morphological characteristics established in studies of rats and mice under basal conditions.3,5,10

Morphometric Analysis

Apical plasma membrane boundary length, cytoplasmic area, and gold label along the apical plasma membrane and over the cytoplasm, including cytoplasmic vesicles, were quantified in type B intercalated cells and non-A, non-B intercalated cells10,20 in 4 to 6 individual mice from each group. A least 5 of each intercalated cell type were selected randomly in each animal and photographed at a primary magnification of 5000× and examined at a final magnification of ≈18 200×. The exact magnification was calculated by using a calibration grid with 1134 lines/mm. Apical plasma membrane boundary length and cytoplasmic area were determined by using point and intersection counting, the Merz curvilinear test grid, and standard stereologic formulas.20

Statistical Analysis

For morphometric data without normal distribution or equal variance, a Mann-Whitney rank-sum test was used. In all other studies, comparisons were made between 2 groups with an unpaired Student t test. A P<0.05 indicates statistical significance. Data are displayed as mean±SEM.

The effect of DOCP expression on Pds mRNA was examined. Pds mRNA/kidney was 4.37±0.38×109 template molecules (n=10) in controls but increased to 7.12±0.87×109 in DOCP-treated mice (n=10, P<0.05). In contrast, no difference in β-actin mRNA/kidney was observed between controls and DOCP-treated mice (1.53±0.13×1011 template molecules in controls, n=10, versus 1.65±0.25×1011 in DOCP-treated mice, n=10). We conclude that DOCP upregulates Pds message expression in kidney.

Effect of DOCP on the Expression and Subcellular Distribution of Pendrin

We tested the effect of DOCP on pendrin protein expression and the subcellular distribution of pendrin. By immunohistochemistry and light microscopy, pendrin labeling was similar in kidneys from control and DOCP-treated mice. In kidneys from both groups, pendrin labeling was observed over the apical region of a subset of cells within the iCT and the CNT (Figures 1a and 1b), as described previously.10,11 Within the CCD, pendrin immunoreactivity was present over the apical region of a subset of cells, as described previously (Figure 2).7,10,11 However, in the apical region of cells within the CCD, pendrin labeling appeared to be more intense and more discrete in kidneys from DOCP-treated mice than in controls (Figures 2a and 2b). Thus, possible DOCP-induced changes in the subcellular distribution of pendrin were studied in more detail.

Figure 1. Effect of DOCP on pendrin expression in the cortical labyrinth. a, Pendrin labeling in the cortical labyrinth from an untreated mouse (Series 1). Distribution of pendrin immunolabel is similar to that reported previously.10 A subpopulation of cells in the iCT and CNT (arrows) exhibits intense apical immunoreactivity for pendrin even under basal conditions. b, Pendrin immunolabeling in a subpopulation of cells (arrows) in the ICT and CNT from a mouse treated with DOCP. By light microscopy, pendrin labeling was similar in the cortical labyrinth of control and DOCP-treated mice.

Figure 2. Effect of DOCP on pendrin expression in medullary rays. a, Pendrin immunolabeling in the CCD from an untreated mouse (series 1). Pendrin labeling was observed in a subset of cells within the CCD, as observed previously. b, Pendrin labeling in the cortical labyrinth from a mouse treated with DOCP. Pendrin immunolabel was present in a subpopulation of cells in the CCD (arrows), as in control animals, but the apical label appeared to be more intense and more discrete in mice treated with DOCP.

In mouse CCDs, we had observed previously that the majority of non-A intercalated cells are type B intercalated cells.5 Thus, pendrin protein expression in the apical plasma membrane and apical cytoplasmic vesicles was quantified in type B intercalated cells from both untreated and DOCP-treated mice by immunogold cytochemistry, transmission electron microscopy, and morphometric analysis. Type B cells typically exhibit a smooth apical plasma membrane surface, numerous cytoplasmic vesicles, a subapical band free of vesicles, and abundant mitochondria (Figure 3a).10 In control animals, the ultrastructural features and distribution of pendrin immunolabel were similar to our previous observations.10 Pendrin immunoreactivity was prevalent over apical cytoplasmic vesicles, but little immunolabel was present along the apical plasma membrane (Figure 3b). However, in DOCP-treated mice, type B intercalated cells typically exhibited a marked increase in apical plasma membrane microprojections and intense pendrin immunolabel along the apical plasma membrane (Figure 4). Morphometric analysis (Table 1) demonstrated more than a 2-fold increase in apical plasma membrane boundary length in type B cells from DOCP-treated mice relative to controls, and a 2-fold increase in pendrin label density along the apical plasma membrane. Thus, a 6-fold increase in pendrin immunolabel along the apical plasma membrane of the type B cell was measured. Moreover, the ratio of immunolabel in the apical plasma membrane to label in the cytoplasm and cytoplasmic vesicles increased 8-fold. However, no significant change in total pendrin labeling in type B intercalated cells was noted in mice treated with DOCP. We conclude that DOCP treatment induces a marked shift in the subcellular distribution of pendrin in the type B intercalated cell of the CCD, resulting in increased expression of pendrin in the apical plasma membrane with little change in total pendrin protein expression per cell.

Figure 3. Transmission electron photomicrograph of type B intercalated cell from an untreated mouse. In control animals, the ultrastructure of the type B intercalated cells was similar to that described under basal conditions.10 As illustrated in panel a, type B intercalated cells typically had a relatively smooth apical plasma membrane, with few microprojections, prominent cytoplasmic vesicles, and a subapical band of vesicle-free cytoplasm. b, Higher magnification of the apical region of the same cells. In control animals, immunogold label for pendrin was primarily located over apical cytoplasmic vesicles (arrowheads), and pendrin immunolabel along the apical plasma membrane was sparse. Magnifications: a, 6600×; b, 9900×.

Figure 4. Transmission electron photomicrograph of type B intercalated cell from mouse treated with DOCP. As illustrated in a, in type B intercalated cells in the CCD of DOCP-treated mice, there was a marked increase in the number and length of apical plasma membrane microprojections compared with type B intercalated cells from control mice. b, Apical region of this same cell at higher magnification. In further contrast to control animals, in DOCP-treated mice, type B intercalated cells exhibited intense immunogold label for pendrin along the apical plasma membrane (arrows) in addition to label over apical cytoplasmic vesicles (arrowheads). Magnifications: a, 6600×; b, 9900×.

The effect of DOCP on the distribution of pendrin in non-A, non-B intercalated cells differed from its effect in type B intercalated cells (Table 1). In non-A, non-B intercalated cells from mice treated with DOCP, boundary length did not change; however, pendrin label in the apical plasma membrane increased 2-fold owing to increased density of pendrin labeling. Moreover, total pendrin label per cell was increased 2-fold in this cell type.

We conclude that pendrin apical plasma membrane label density is increased by DOCP treatment to a similar extent in type B intercalated cells and in non-A, non-B intercalated cells. However, pendrin expression in the apical plasma membrane is increased to a greater extent in type B intercalated cells than in non-A, non-B cells because of the marked increase in apical plasma membrane boundary length that occurs in the type B cell after administration of this aldosterone analogue.

Effect of DOCP on Pds (+/+) and Pds(-/-) Mice

Because pendrin is upregulated with DOCP, further studies investigated whether a renal phenotype is unmasked in Pds(-/-) mice treated with aldosterone analogues (Figures 5 and 6⇓ and Table 2). In the absence of DOCP, after 7 days of paired intake of the gelled diet, systolic blood pressure was similar in Pds(-/-) and Pds (+/+) mice. Moreover, weight was unchanged in both groups over the treatment period (Figure 5). When mice were treated with DOCP and then ate the gelled diet for 6 days, wild-type mice gained weight and became hypertensive, whereas Pds(-/-) mice treated with DOCP had no change in blood pressure and did not gain weight, despite an identical intake of the diet during this period (79.2±2.9 g, Pds (+/+); 78.8±2.3 g, Pds(-/-); P=NS, Figure 5). Table 2 shows that serum electrolytes and 24-hour urinary NaCl excretion were the same in wild-type and Pds(-/-) mice 7 days after administration of DOCP. Thus, pendrin is critical in the process of DOCP-induced hypertension and weight gain.

Figure 5. Effect of DOCP on body weight and systolic blood pressure in Pds (-/-) and Pds (+/+) mice. All mice were pair-fed a balanced, high-salt, gelled diet. Some mice were treated with DOCP. After 7 days of diet alone or diet plus DOCP, systolic blood pressure and weight change for the treatment period were measured.

Figure 6. Effect of DOCP on arterial pH, Pco2, and HCO3− in Pds (-/-) and Pds (+/+) mice. Mice were treated as described in the legend to Figure 5. After 7 days of diet alone or diet plus DOCP, arterial blood gases were measured.

TABLE 2. Serum and Urine Chemistries in Pds (−/−) and (+/+) Mice Taken 7 Days After DOCP Treatment

Because pendrin transports HCO3−,8 arterial blood gases were measured in Pds (-/-) and (+/+) mice. In the absence of DOCP, arterial pH and calculated HCO3− were similar in both groups. After DOCP treatment, both Pds (+/+) and Pds (-/-) mice developed metabolic alkalosis. However, the metabolic alkalosis was more severe in DOCP-treated Pds (-/-) mice. Seven days after DOCP treatment, Pds (-/-) mice had a lower urinary pH than did wild-type mice, although net acid excreted over 24 hours was similar. We conclude that pendrin attenuates DOCP-induced metabolic alkalosis.

Discussion

Aldosterone is critical to the regulation of both net acid secretion and absorption of NaCl along the collecting duct. In the CCD, aldosterone administration in vivo leads to increased secretion of HCO3− and K+. Aldosterone administration in vivo also increases absorption of NaCl through upregulation of the Na+ channel ENaC,23 the thiazide-sensitive NaCl cotransporter,24 and the Na,K-ATPase,25 which causes Na+ retention and increased blood pressure. Which renal Cl− transporters are regulated by aldosterone is poorly understood. Moreover, the extent to which aldosterone-induced hypertension occurs through changes in Cl− transporter expression in the kidney is unknown.26 To our knowledge, this study is the first to show a role for a Cl− transporter in aldosterone-induced hypertension.

Administration of aldosterone in vivo induces a dramatic increase in both Cl− absorption and HCO3− secretion in the CCD. After DOC administration in vivo, the Cl− concentration in the collected luminal fluid of rabbit CCD drops to 89 mEq/L from perfusate values of 112 mEq/L,27 whereas collected HCO3− concentration increases from 25 to >40 mmol/L.15 Most transepithelial Cl− transport in this segment occurs either through paracellular transport or through transepithelial transport across intercalated cells, rather than across principal cells.28 Across intercalated cells, aldosterone-stimulated, active Cl− absorption occurs through apical Cl−/HCO3− exchange in series with a basolateral Cl− conductance.4

There is growing evidence that pendrin contributes to the apical anion exchange process of the type B intercalated cell. Both pendrin and the putative apical anion exchanger of the type B intercalated cell transport Cl− and HCO3−.8,29 Both apical anion-exchange activity and pendrin protein expression are downregulated in models of metabolic acidosis, eg, with NH4Cl ingestion,12–14,30,31 and are upregulated in models of metabolic alkalosis, eg, with NaHCO3 ingestion.12–14 Apical anion exchange in the CCD is also upregulated by DOCP.15,16,32,33 This study is the first to demonstrate regulation of pendrin by an aldosterone analogue. After DOCP treatment, we observed a 6-fold increase in pendrin immunoreactivity in the apical plasma membrane of the type B intercalated cell and a 2-fold increase in the apical plasma membrane of the non-A, non-B intercalated cell. This observation is consistent with previous reports that demonstrated greater change in pendrin expression in the type B intercalated cell than in the non-A, non-B cell with changes in acid-base balance.14

Aldosterone administered in vivo is a well-established model of hypokalemic metabolic alkalosis. Thus, DOCP might upregulate pendrin indirectly through hypokalemia and/or metabolic alkalosis. However, in another model of hypokalemic metabolic alkalosis (dietary K+ restriction), pendrin expression is reduced in the apical plasma membrane.13 Thus, pendrin is not upregulated in all models of hypokalemic metabolic alkalosis. However, whether the effect of DOCP pendrin expression is a direct effect of the hormone or an indirect effect, such as through changes in vascular volume, will require examination of other treatment models.

Apical Na+-independent Cl−/HCO3− exchange of the type B cell and pendrin participate in secretion of OH− equivalents during metabolic alkalosis.7,33 In mice treated with DOCP and ingesting NaHCO3, secretion of HCO3− in the CCD is Pds dependent.7 During metabolic alkalosis, CCD from wild-type, Pds (+/+) mice secrete HCO3−.7 However, CCDs from Pds (-/-) mice absorbed HCO3− when studied under the same treatment conditions.7 In the present study, we observed that after DOCP treatment, Pds (-/-) mice had a more severe metabolic alkalosis than did wild-type mice, consistent with the defect in secretion of OH− equivalents demonstrated previously in the CCD of Pds (-/-) mice.7

We observed that Pds (+/+) mice treated with DOCP gained weight and developed hypertension. However, pair-fed, DOCP-treated Pds (-/-) mice did not become hypertensive and did not gain weight. This DOCP resistance likely occurred because of the inability of the DCT, CNT, and CCD of Pds (-/-) mice to absorb Cl− fully. However, this hypothesis requires further study. Abnormalities in fluid absorption and secretion have been observed in other organ systems with disruption of the Pds gene. Patients with the Pendred syndrome and Pds-knockout mice have deafness and structural abnormalities of the inner ear, including endolymphatic hydrops.17 In both the inner ear and kidney, pendrin might participate in the absorption of Cl−. The functional absence of pendrin might lead to loss of Cl− in the urine and retention of Cl− in the endolymphatic fluid of the ear. The endolymphatic sac of the ear contains mitochondria-rich, type A–like cells that express the H-ATPase on the apical plasma membrane and type B–like cells that express pendrin on the apical plasma membrane.34 Thus, cells of the endolymphatic sac resemble renal intercalated cells both ultrastructurally and functionally.34

It is likely that with 7 days of DOCP treatment, the cumulative excretion of NaCl and net acid differ in Pds (+/+) and (-/-) mice. However, because 24-hour urinary NaCl and net acid excretion did not differ between the 2 groups of mice after 7 days of DOCP treatment, differences in excretion most likely occurred within the first 6 days of DOCP administration.

Perspectives

The present study is the first to demonstrate a role for pendrin in mineralocorticoid-induced hypertension. Moreover, this study raises the possibility that pendrin could be the target of future antihypertensive and/or diuretic drugs.

Acknowledgments

This work was supported by the National Institute of Digestive, Diabetes and Kidney Diseases grant DK 52935 (to S.M. Wall). We thank Mae De La Calzada, Department of Medicine, University of Texas Medical School, Houston, and Melissa A. Lewis, Sharon W. Matthews, and John C. Bitter, University of Florida College of Medicine Electron Microscopy Core Facility, Gainesville, Fla, for their technical assistance. We thank Dr Greg Shipley, Department of Integrative Biology, UTHMC, for his suggestions, and Dr. Robert Beach, Department of Medicine, University of Texas Medical Branch, Galveston, Texas, for his assistance.